Method for producing hydrogel or dried hydrogel, hydrogel or dried hydrogel, collagen pathological model or collagen aging model, and cultured meat

Abstract

Provided is a method for producing a hydrogel or dried hydrogel having a desired shape, the method having: a step 1 for injecting a colloid solution into a container having a prescribed shape, at least a part of which has a separation membrane; and a step 2 for placing the container in a liquid phase or in a gas phase and gelling the colloid solution in the container to obtain a hydrogel.

Description

Method for producing hydrogel or dried hydrogel, hydrogel or dried hydrogel, collagen pathological model or collagen aging model, and cultured meat

[0001] The present invention relates to a method for producing a hydrogel or a dried hydrogel, a hydrogel or a dried hydrogel, a collagen pathological model or a collagen aging model, and cultured meat.

[0002] The present inventors have previously developed drug discovery support tools and medical devices using collagen vitrigel (see, for example, Patent Documents 1 and 2). Regarding drug discovery support tools, they have pioneered the development of an ocular irritation test method and a microvascular permeability test method, respectively, using the time-dependent changes in transepithelial and transendothelial electrical resistance of barrier tissue reconstructed from cultured cells as indicators (see, for example, Patent Document 1). Regarding medical devices, they have developed devices (atelocollagen vitrigel processed into various shapes) that inhibit scarring or stenosis and promote epithelialization (see, for example, Patent Document 2).

[0003] International Publication No. 2021 / 075451 International Publication No. 2022 / 085377

[0004] However, preparation of collagen vitrigel remains difficult. In conventional methods for producing collagen vitrigel, a collagen sol must be prepared by uniformly mixing an acid-soluble collagen solution with a gelling agent such as a culture medium. There are limitations to the final concentration of collagen sol that can be prepared; for example, the final concentration must be 0.25 (w / v)% or less for native collagen and 0.50 (w / v)% or less for atelocollagen, and the volume of each sol must be 50 mL or less, otherwise uniform mixing is extremely difficult.

[0005] Furthermore, since collagen vitrigel is produced by gelling, vitrifying, and rehydrating a prepared collagen sol, it takes a long time, more than two weeks, to produce it. Furthermore, the limit of collagen concentration in collagen vitrigel is approximately 30 (w / v)%, and it has not been possible to produce collagen gels of any desired concentration.

[0006] The present invention has been made in consideration of the above circumstances, and provides a fundamental technology for producing collagen vitrigel, a new material of high-density collagen fiber networks, by drastically improving the method for producing collagen vitrigel, a gel of any concentration and shape, a gel of collagen containing a limited molecular weight fraction component in any solution, and a fundamental technology for producing said gel. That is, the present invention provides a fundamental technology for producing a gel with adjusted concentration, ingredients, and shape (Concentration, Ingredient, and Shape-adjusted Gel; abbreviated as "CIS Gel").

[0007] The present invention includes the following aspects: [1] A method for producing a hydrogel or a dried hydrogel having a desired shape, comprising: Step 1: pouring a colloidal solution into a container of a predetermined shape having a separation membrane at least partially therein; and Step 2: placing the container in a liquid phase or a gas phase and gelling the colloidal solution in the container to obtain a hydrogel. [2] The method according to [1], further comprising: Step 3: adjusting the concentration of the colloidal solution by evaporating water from the colloidal solution through the separation membrane in the container in the gas phase between Steps 1 and 2. [3] The method according to [1], further comprising: Step 3: adjusting the concentration of the colloidal solution by dehydrating water from the colloidal solution through the separation membrane by immersing at least a portion of the separation membrane of the container in a liquid phase containing salt. [4] The method according to [1], wherein the separation membrane is a dialysis membrane, an ultrafiltration membrane, a microfiltration membrane, a collagen vitrigel membrane, or a food packaging casing. [5] The manufacturing method according to [1], in which an insoluble substance of any shape is placed in the colloidal solution and then gelled to prepare a hydrogel containing the insoluble substance. [6] The manufacturing method according to [5], in which the insoluble substance is leather or crushed hide from a leather manufacturing process. [7] The manufacturing method according to [5], in which the shape of the hydrogel is induced by removing the insoluble substance after gelation. [8] The manufacturing method according to [1], in which gel formation is induced by allowing a gelling agent in the liquid phase to diffuse, penetrate, or permeate through the separation membrane. [9] The manufacturing method according to [1], in which gel formation is induced by a temperature change in the liquid or gas phase.

[10] The manufacturing method according to [1], in which the separation membrane is a dialysis membrane, ultrafiltration membrane, or microfiltration membrane, and has pores that allow molecules of an allowable molecular weight or less to diffuse, penetrate, or permeate, and in step 2, a limited molecular weight fraction component in the liquid phase is allowed to diffuse, penetrate, or permeate into the colloidal solution or the hydrogel through the pores.

[11] The method according to [1], further comprising step 4 of concentrating the concentration of the hydrogel or obtaining a dried hydrogel by placing the container in a gas phase and evaporating the water content of the hydrogel obtained in step 2 through the separation membrane.

[12] The method according to any one of [1] to

[11] , wherein the container of a predetermined shape is a culture insert-type container.

[13] A method for producing a hydrogel or dried hydrogel of a desired shape, comprising: Step 5: injecting a gelling agent solution into a first container of a predetermined shape having a separation membrane at least in a portion thereof; and Step 6: injecting a colloidal solution into a second container of a predetermined shape, and then contacting the gelling agent solution with the colloidal solution in the second container through the separation membrane in the first container to gel the colloidal solution, thereby obtaining a hydrogel.

[14] The production method according to

[13] , comprising Step 7: adjusting the concentration of the colloidal solution by evaporating water in the colloidal solution in the second container in a gas phase between Steps 5 and 6.

[15] A hydrogel or dried hydrogel having uniformly colloidal particles and limited molecular weight fraction components.

[16] The hydrogel or dried hydrogel according to

[15] , which is a collagen gel, a gelatin gel, a hen's egg-derived gel, or an agarose gel.

[17] The hydrogel or dried hydrogel according to

[15] , wherein the limited molecular weight fraction component comprises a component derived from a cell culture medium, a component derived from a conditioned culture medium, a component derived from a body fluid, a food component, or an active cosmetic ingredient.

[18] A collagen pathology model or a collagen aging model uniformly containing collagen and advanced glycation end products.

[19] Cultured meat comprising a culture of a hydrate of a hydrogel or a dried hydrogel and cells from an edible part of an animal.

[0008] According to the present invention, gels of collagen or the like of any concentration, including high concentrations, can be easily prepared in a short time without necessarily requiring vitrification and rehydration steps, and hydrogels containing limited molecular weight fraction components in any solution can be easily prepared.

[0009] FIG. 1 is a perspective view showing an example of a culture insert-type container. FIG. 2 is a photograph showing the natural concentration process of a native collagen solution observed over time in a refrigerator. FIG. 3 is a photograph showing the preparation process of a collagen sol and the progress of gelation observed over time in a refrigerator. FIG. 4 is a photograph showing the production process of a dried collagen gel and a collagen vitrigel-like gel obtained by rehydrating the dried collagen gel over time. FIG. 5 is a photograph showing the production process of a udon-shaped boiled egg white by concentrating chicken egg white and then boiling it in hot water. FIG. 6 is a photograph showing the handling properties of soft, cylindrical boiled egg white obtained by boiling chicken egg white without concentrating it. FIG. 7 is a photograph showing the production process of kishimen-like boiled egg white over time. FIG. 8 is a photograph showing the production process of a plastic rod-containing collagen vitrigel-like gel or a tubular collagen vitrigel-like gel and a mesh-containing collagen vitrigel-like gel over time. FIG. 9 is a photograph showing the production process of a udon-shaped egg white gel observed over time. Photographs showing the time course of the production process of regenerated leather (dried native collagen vitrigel-like gel containing pulverized leather and dried atelocollagen vitrigel-like gel containing pulverized leather). Photographs showing the time course of the production process of a membranous atelocollagen vitrigel-like gel dried body. Photographs showing the time course of the production process of a cylindrically hollowed collagen gel. Photographs showing the time course of the production process of an insert-type 0.5% collagen gel-layered regenerated cellulose membrane culture carrier. Photographs showing the time course of the production process of an insert-type 0.5% collagen gel-layered collagen vitrigel membrane culture carrier. Photographs showing the time course of the production process of a 12-well plate-type 0.5% collagen gel culture carrier. Photographs showing the time course of observation with a phase contrast microscope after seeding C2C12 cells on an insert-type 0.5% collagen gel-layered regenerated cellulose membrane culture carrier and initiating culture. 1 shows photographs of C2C12 cells cultured for 7 days on an insert-type 0.5% collagen gel-layered collagen vitrigel membrane culture carrier glycated at different glucose concentrations, followed by observation with a phase-contrast microscope. 1 shows photographs of C2C12 cells cultured over time on a 12-well plate-type 0.5% collagen gel culture carrier after initiating culture.

[0010] Hereinafter, embodiments of the present invention will be described in detail. <<Method for Producing a Hydrogel or a Dried Hydrogel>> [First Embodiment] In one embodiment, the present invention provides a method for producing a hydrogel or a dried hydrogel having a desired shape, the method comprising: Step 1 of pouring a colloidal solution into a container of a predetermined shape having an ultrafiltration membrane or a microfiltration membrane at least in a portion thereof; and Step 2 of placing the container in a liquid phase or a gas phase and gelling the colloidal solution in the container to obtain a hydrogel.

[0011] <Step 1> Step 1 involves pouring a colloidal solution into a container of a predetermined shape that at least partially contains a separation membrane. Separation membranes are typically selected based on the size of the substances to be separated. For example, reverse osmosis membranes are used for target substances (monovalent ions, various molecules, etc.) that are approximately 1 nanometer or less in size (molecular weight: approximately 100 or less), nanofiltration membranes are used for target substances (polyvalent ions, low-molecular weight compounds, etc.) that are approximately 1 to 2 nanometers in size (molecular weight: approximately 200 to 1,000), dialysis membranes and ultrafiltration membranes are used for target substances (proteins, macromolecules, viruses, etc.) that are approximately 2 to 100 nanometers in size (molecular weight: approximately 1,000 or more), microfiltration membranes are used for target substances (colloids, bacteria, etc.) that are approximately 100 nanometers to 10 micrometers in size, and prefilters are used for target substances (visible particles, etc.) that are approximately several micrometers or larger in size. Reverse osmosis membranes, nanofiltration membranes, dialysis membranes, ultrafiltration membranes, and microfiltration membranes are membranes with pores that allow only molecules or ions with a certain molecular weight or less to pass through. For example, when a container having at least a dialysis membrane, ultrafiltration membrane, or microfiltration membrane is immersed in a liquid phase, the dialysis membrane, ultrafiltration membrane, or microfiltration membrane prevents colloidal particles in a colloidal solution from passing through to the outside of the container, while allowing, for example, low molecular weight components dissolved in a culture medium to pass through to the inside of the container. More specifically, the dialysis membrane, ultrafiltration membrane, or microfiltration membrane may be permeable to, for example, high molecular weight compounds with a molecular weight of about 100,000 or less. For example, it may be permeable to molecular compounds with a molecular weight of about 10,000 or less, or to low molecular weight compounds with a molecular weight of several thousand or less. Furthermore, it may be a collagen vitrigel membrane or a food packing casing that has the same permeability as the dialysis membrane, ultrafiltration membrane, or microfiltration membrane. The term "vitrigel" refers to a gel in a stable state obtained by vitrifying a conventional hydrogel, in which free water in the hydrogel is completely removed and then bound water is partially removed, followed by rehydration, and the present inventors have named this gel "vitrigel (registered trademark)." In addition, when using the term "vitrigel" in this specification, the term "(registered trademark)" may be omitted.

[0012] Furthermore, for example, when a container having at least a part thereof a sterile filtration filter that is a microfiltration membrane is immersed in a liquid phase, the sterile filtration filter does not allow colloid particles in a colloidal solution to pass to the outside of the container, while allowing, for example, a molecular weight cutoff component dissolved in a culture solution to pass to the inside of the container. More specifically, the sterile filtration filter may be, for example, any of various membrane filters that can pass particles with a diameter of 0.45 μm or less, and may also be, for example, any of various membrane filters that can pass particles with a diameter of 0.22 μm or less.

[0013] A colloidal solution refers to a solution in which dispersoid colloidal particles (size: approximately 1 to several hundred nm) in a liquid as a dispersion medium are composed, in particular, of polymeric compounds. More specific examples of colloidal solutions include aqueous solutions of natural polymeric compounds and synthetic polymeric compounds. When these polymeric compounds are crosslinked by chemical bonding to form a network structure, they transition to a "hydrogel," a semi-solid substance that retains a large amount of water in the network. In other words, a "hydrogel" refers to a colloidal solution that has been gelled.

[0014] The colloidal solution used as the raw material for the hydrogel may be any biocompatible material, and examples thereof include gelling extracellular matrix-derived components, natural polymer compounds such as fibrin, agar, agarose, and cellulose, and synthetic polymer compounds such as polyacrylamide, polyvinyl alcohol, polyethylene oxide, and poly(II-hydroxyethylmethacrylate) / polycaprolactone.

[0015] Examples of gelling extracellular matrix-derived components include, but are not limited to, collagen (type I, type II, type III, type V, type XI, etc.), basement membrane components reconstituted from mouse EHS tumor extract (including type IV collagen, laminin, heparan sulfate proteoglycan, etc.) (trade name: Matrigel), glycosaminoglycans, hyaluronic acid, proteoglycans, gelatin, etc. The desired hydrogel can be produced by selecting the optimal salts and other components for gelation, their concentrations, pH, etc. Furthermore, by combining raw materials, hydrogels that mimic various in vivo tissues can be obtained.

[0016] Among these, gelling extracellular matrix-derived components are preferred for the colloidal solution, with collagen being more preferred. Among collagens, native collagen or atelocollagen are preferred raw materials, with atelocollagen from which antigenic telopeptides have been removed being even more preferred for transplantation into living organisms. The grade of collagen to be used is selected depending on the field of use. Specifically, medical grade is used in the medical field; research reagent grade is used in in vitro assay systems for drug discovery support; crude collagen extracted from livestock is used in the food field if food safety can be confirmed; and crude collagen extracted from various animals is used in the regenerated leather field if environmental safety can be confirmed.

[0017] Alternatively, edible colloidal solutions may be used, such as those containing egg white (containing ovalbumin as a main component), collagen, gelatin, and agarose.

[0018] The container of a predetermined shape may be a dialysis tube or a culture insert-type container. FIG. 1 is a perspective view showing an example of a culture insert-type container. The culture insert-type container 10 shown in FIG. 1 comprises a cylindrical portion 1, a flange portion 2, and an ultrafiltration membrane or microfiltration membrane 3. The cylindrical portion 1 and the flange portion 2 are preferably made of acrylic or polystyrene. The culture insert-type container holding the colloidal solution can be easily operated by leaning it against, for example, a culture dish holding a culture medium. According to this embodiment, a hydrogel or dried hydrogel of a desired shape can be produced by changing the shape of the container.

[0019] <Step 2> Step 2 involves placing a container of a predetermined shape in a liquid or gas phase and gelling the colloidal solution in the container to obtain a hydrogel. The liquid phase is not particularly limited as long as it has the ability to gel the colloidal solution. A liquid containing a predetermined molecular weight cutoff component that can permeate the pores of the separation membrane is preferred, and a culture medium or salt solution containing a gelling agent is more preferred. The salt solution may be an aqueous solution of inorganic salts, as well as natural seawater or a diluted solution of seawater. The separation membrane has pores that allow molecules with a molecular weight below the allowable range to diffuse, penetrate, or permeate, allowing the limited molecular weight fraction component in the liquid phase to diffuse, penetrate, or permeate into the colloidal solution or hydrogel through the pores. When the liquid phase contains a gelling agent, gel formation is induced by diffusing, penetrating, or permeating the gelling agent in the liquid phase. It is preferable to stir the liquid phase using a stirrer. For example, collagen gels are prone to clumping and are difficult to form uniformly. However, diffusing and penetrating a gelling agent in the liquid phase allows a hydrogel in which colloidal particles are uniformly dispersed to be obtained.

[0020] The liquid phase may also be a liquid at a predetermined temperature. By using a liquid at a predetermined temperature as the liquid phase, gel formation is induced by a temperature change in the liquid phase. For example, when egg white is used as the colloidal solution, placing it in hot water at 60°C or higher will cause denaturation and induce gel formation. To add flavor to jelly-like or solidified egg white, seasoning ingredients such as salt and sugar can be added to the liquid phase. Furthermore, by using soy sauce desalted using a reverse osmosis membrane or the like as the liquid phase, it is possible to impart flavor without containing the salt and other low-molecular-weight components in soy sauce. The liquid at a predetermined temperature used as the liquid phase is not particularly limited, and examples include sterilized water, physiological saline, PBS, etc. Furthermore, the gas phase may be a space with controlled temperature and humidity. For example, when a gelatin aqueous solution is used as the colloidal solution, gel formation is induced by placing it at room temperature or below, preferably in a refrigerator.

[0021] Alternatively, a gel containing an insoluble substance may be prepared by placing an insoluble substance of any shape in a colloidal solution and then gelling it. Examples of insoluble substances include, but are not limited to, substances that impart a specific shape to the gel or substances that impart specific physical properties to the gel. Examples of substances that impart a specific shape to the gel include, but are not limited to, plastics of a predetermined shape. Examples of substances that impart specific physical properties to the gel include, for example, fillers. By including a filler in the gel, high strength can be achieved. In particular, by drying a collagen gel densely containing leather or crushed hides from the leather manufacturing process as a filler and irradiating it with ultraviolet light, it becomes possible to produce plastic-free recycled leather. A core material may also be used to impart specific physical properties to the gel. For example, when the gel is filamentous, the inclusion of a filamentous core material can improve breaking strength. Examples of filamentous core materials include chemical fibers such as nylon yarn, polyester yarn, rayon yarn, and polyglactin yarn, as well as natural yarns such as silk yarn, cotton yarn, linen yarn, and wool yarn.

[0022] Furthermore, the shape of the gel may be induced by removing the insoluble substance after gelation. A cylindrical plastic may be used as an example to give the gel a specific shape. A cylindrical plastic is placed in the center of a container, and removed after gelation to obtain a hollow gel (so-called "chikuwa"-shaped).

[0023] <Step 3> Furthermore, between step 1 and step 2, step 3 may be included in which the concentration is adjusted by evaporating the water in the colloidal solution through the membrane in the container in the gas phase. In conventional methods for producing collagen vitrigel, it was extremely difficult to uniformly mix the gelling agent to prepare a collagen sol unless the final collagen concentration was a predetermined concentration or less. According to this embodiment, a hydrogel of any concentration can be produced.

[0024] Various methods can be used to evaporate the water in the colloidal solution, such as air drying, drying in a sealed container (air is circulated in the container to constantly supply dry air), drying in an environment with silica gel placed, etc. For example, examples of the air drying method include drying for two days in an incubator kept sterile at 10°C and 40% humidity, or drying at room temperature for one day and night in a sterile clean bench.

[0025] For example, when egg white is used as the colloidal solution, the water content in the egg white is evaporated from the container in the gas phase, and then the container is placed in hot water at 60°C or higher to induce gel formation through denaturation, thereby adjusting the hardness to an appropriate level.

[0026] A step 3 of adjusting the concentration by dehydrating the water in the colloidal solution through the membrane in the container in a liquid phase containing a salt may be included between the steps 1 and 2. By utilizing the phenomenon of water moving from a compartment with a high osmotic pressure to a compartment with a low osmotic pressure and using a liquid phase containing a salt, the water concentration in the colloidal solution can be adjusted even in the liquid phase.

[0027] <Step 4> The method may further include step 4, in which the container is placed in a gas phase and the water content of the hydrogel obtained in step 2 is evaporated through the membrane to concentrate the concentration of the hydrogel or to obtain a dried hydrogel. A preferred method for evaporating the water content is the same as that in step 3. For example, when egg white is used as the colloidal solution, the solution can be placed in hot water at 60°C or higher to induce gel formation by denaturation, and then the water content can be evaporated in the gas phase to adjust the solution to an appropriate hardness.

[0028] Rehydration may be performed after step 4. When the hydrogel is a collagen gel, a collagen vitrigel-like gel is obtained by this step. Alternatively, the collagen vitrigel-like gel may be re-dried (re-vitrified) to obtain a dried collagen vitrigel-like gel.

[0029] The term "vitrigel" refers to a stable gel obtained by vitrifying a conventional hydrogel and then rehydrating it, and has been named "vitrigel (registered trademark)" by the present inventors. Here, the hydrogel (CIS gel) with the concentration, components, and shape adjusted according to the present invention is different from conventional hydrogels, and therefore the hydrogel obtained by drying the hydrogel prepared according to the present invention and then rehydrating the dried hydrogel is referred to as a vitrigel-like gel. Furthermore, when using the term "vitrigel" in this specification, the term "(registered trademark)" may be omitted.

[0030] After step 4, the dried hydrogel may be irradiated with ultraviolet light. By irradiating with ultraviolet light, crosslinking structures are formed between and within the collagen molecules that constitute the vitrigel, thereby increasing the strength of the vitrigel. The irradiation energy of the ultraviolet light may be adjusted appropriately depending on the composition and content of the dried hydrogel. The irradiation energy of the ultraviolet light may be, for example, 0.1 mJ / cm. ２ More than 6000mJ / cm ２ It is sufficient if it is less than, for example, 10 mJ / cm ２ More than 4000mJ / cm ２ It is sufficient if it is less than, for example, 20 mJ / cm ２ More than 500mJ / cm２ The following may be used: Aqueous solutions used for rehydration include sterilized water, physiological saline, PBS, etc.

[0031] [Second Embodiment] In one embodiment, the present invention provides a method for producing a hydrogel or a dried hydrogel having a desired shape, the method comprising: step 5 of injecting a gelling agent solution into a first container of a predetermined shape having a separation membrane at least in a portion thereof; and step 6 of injecting a colloidal solution into a second container of a predetermined shape, and then bringing the gelling agent solution into contact with the colloidal solution in the second container through the separation membrane in the first container, thereby gelling the colloidal solution to obtain a hydrogel.

[0032] <Step 5> Step 5 is a step of injecting a gelling agent solution into a first container of a predetermined shape having a separation membrane at least in a portion thereof. The first container of a predetermined shape may be, for example, a culture insert-type container as shown in Figure 1. Examples of the separation membrane include those similar to those described in Step 1.

[0033] <Step 6> Step 6 is a step of injecting a colloidal solution into a second container of a predetermined shape, and then contacting the gelling agent solution with the colloidal solution in the second container through the separation membrane in the first container to gel the colloidal solution and obtain a hydrogel. The second container of a predetermined shape is not particularly limited as long as it allows the injected colloidal solution to come into contact with the separation membrane of the first container, and examples include culture dishes. For example, operation can be simplified by leaning a culture insert-type container holding the gelling agent solution against a culture dish holding the colloidal solution.

[0034] <Step 7> Step 7 may be included between step 5 and step 6, in which the concentration is adjusted by evaporating the water in the colloidal solution in the second container in the gas phase. If the culture dish holding the colloidal solution is left in the gas phase, the concentration is automatically adjusted by evaporation of the water.

[0035] Thus, a hydrogel is formed in the second container. According to this embodiment, for example, by using a collagen solution as the colloidal solution, a collagen gel culture carrier of a desired concentration can be prepared in the culture dish. Furthermore, by adding glucose or fructose to the gelling agent, a glycated collagen gel culture carrier can be prepared in the culture dish.

[0036] <Hydrogel or Dried Hydrogel> In one embodiment, the present invention provides a hydrogel or dried hydrogel having a uniform distribution of colloidal particles and a limited molecular weight fraction component. Examples of a method for producing the hydrogel or dried hydrogel of this embodiment include, but are not limited to, the method described above in <<Method for Producing a Hydrogel or Dried Hydrogel>>. The hydrogel is preferably one of those described in <<Method for Producing a Hydrogel or Dried Hydrogel>>, and is preferably a collagen gel, a gelatin gel, a gel of egg white (containing ovalbumin as a main component) derived from a hen's egg, or an agarose gel.

[0037] The limited molecular weight fraction component may be any desired component to be incorporated into the hydrogel, and preferably includes, for example, a cell culture medium-derived component, a conditioned culture medium-derived component, a body fluid-derived component, a food component, or a cosmetic active ingredient.

[0038] Any cell culture medium commonly used for culturing cells can be used without any problems, and examples thereof include Dulbecco's Modified Eagle Medium (DMEM), Minimum Essential Medium (MEM), Iscove's Modified Dulbecco's Medium (IMDM), and Glasgow's Minimum Essential Medium (GMEM). Examples of components derived from cell culture medium include gelling agents (components containing crosslinkers and salts at a specific pH in the culture medium); inorganic salts such as sodium ions, potassium ions, and calcium ions; carbohydrates such as glucose, galactose, maltose, and fructose; vitamins such as vitamin A, vitamin B, vitamin E, riboflavin, thiamine, and biotin; proteins or peptides such as albumin, transferrin, fibronectin, and fetuin; fatty acids or lipids such as cholesterol and steroids; trace elements such as zinc, copper, selenium, and tricarboxylic acid intermediates; and serum components such as albumin, growth factors, and growth inhibitors.

[0039] Conditioned culture medium refers to a culture medium containing components secreted by the cultured cells themselves during cell culture. For example, conditioned culture medium can be obtained by co-culturing multiple cells with different properties. Examples of components derived from conditioned culture medium include growth factors, substances effective for cell adhesion, and substances that induce cell differentiation.

[0040] Examples of body fluids include blood, plasma, serum, lymph, tissue fluid, urine, saliva, cerebrospinal fluid, nasal secretion, semen, etc. For example, by using a patient's blood-derived component or urine-derived component as the limited molecular weight fraction component, a hydrogel that reflects the patient's pathological condition can be prepared.

[0041] Food ingredients include salt; soy sauce; sugar; amino acids such as glutamic acid, inosinic acid, and guanylic acid; artificial sweeteners; soup stock; food additives such as preservatives, flavorings, and colorings; spices; starch; vegetable protein; and pH adjusters such as sodium acetate. Active cosmetic ingredients include antioxidants such as ascorbic acid, tocopherol, coenzyme Q10, resveratrol, and glutathione; whitening agents such as glabridin and ammonium persulfate; exfoliants such as glycolic acid, malic acid, and mandelic acid; antiseborrheic agents such as sebacic acid and azelaic acid; antisebum agents such as charcoal powder; antibacterial agents such as climbazole and piroctone olamine; antiperspirants such as aluminum chlorohydrate and aluminum sesquichlorohydrate; astringents such as Citrus aurantifolia (lime) flower extract and calcium lactate; makeup removers such as sodium cocoyl glutamate; deodorants such as triethyl citrate and zinc ricinoleate; flavoring agents such as citral and honey; fragrances such as d,l-limonene and coumarin; glycerin, propanediol, and the like. humectants such as propanediol; keratolytic agents such as chloroacetic acid and salicylic acid; moisturizers such as aloe arborescens leaf extract; fragrances such as geraniol and linalool; emollients such as triolein and squalene; refreshing agents such as menthol and menthyl lactate; skin moisturizers such as panthenol and allantoin; skin protectants such as sphingolipids and zinc oxide; smoothing agents such as castor seed oil; soothing agents such as witch hazel extract, chamomile extract, and bisabolol; tonic agents such as arnica montana and capsicum annuum extract; and ultraviolet filters such as methylene bis-benzotriazolyl tetramethylbutylphenol, ethylhexyl methoxycinnamate, caffeine, theine, theobromine, and theophylline. For example, when producing a hydrogel from egg white (containing ovalbumin as a main component) derived from chicken eggs, a dialysis membrane that blocks salt and allows only the flavor components of the soup stock to pass through, or a food packaging casing with the same permeability as the dialysis membrane, can be used to provide a chicken egg-derived gel suitable for consumers with kidney disease.

[0042] In this embodiment, "having uniformly colloidal particles and limited molecular weight fraction components" refers to a state in which the colloidal particles or limited molecular weight fraction components are not aggregated or in contact with each other, but are spaced apart by a certain distance.

[0043] <Applications> The hydrogel of the present embodiment can be easily prepared to contain any limited molecular weight fraction component in a solution, and therefore, by changing the limited molecular weight fraction component, the hydrogel can be suitably used in the medical field, drug discovery field, food field, etc.

[0044] <Collagen Pathological Model or Collagen Aging Model> In one embodiment, the present invention provides a collagen pathological model or collagen aging model that uniformly contains collagen and advanced glycation end products. It is known that the accumulation of advanced glycation end products (AGEs) in collagen increases with aging. AGEs are thought to be involved in aging phenomena such as loss of skin elasticity, stiffening of joints, and hardening of blood vessels. It has also been reported that the amount of AGEs accumulated in skin collagen is higher in diabetic patients compared to healthy individuals. The collagen pathological model of this embodiment reflects the accumulation of AGEs in collagen. According to this embodiment, a pathological model or aging model can be created using only collagen without using cells.

[0045] The method for producing the collagen pathological model or collagen aging model of this embodiment is not limited to, but includes, for example, the above-mentioned <<Method for producing a hydrogel or a dried hydrogel>>. Using a liquid phase containing a predetermined concentration of sugar, collagen is glycated by diffusion and penetration, resulting in the accumulation of AGEs in the collagen. Examples of collagen include native collagen and atelocollagen. Examples of sugar include glucose and fructose. The sugar concentration contained in the liquid phase is preferably 20 mM to 1 M, more preferably 50 mM to 500 mM, and even more preferably 100 mM to 300 mM.

[0046] The collagen pathological model or collagen aging model of this embodiment can be used to screen anti-glycation agents. Using the collagen pathological model of this embodiment, the strength of the inhibitory effect of a test substance on collagen glycation reaction can be evaluated by monitoring the fluorescence (Ex / Em: 370 / 440 nm) generated from glycated collagen. The smaller the Ex / Em value, the stronger the anti-glycation effect is evaluated to be. Evaluation can be easily performed without staining glycated collagen. The anti-glycation agents obtained by screening can be used, by virtue of their action, as preventive or ameliorative agents for wrinkles, sagging, arteriosclerosis, diabetes, osteoporosis, cataracts, etc.; or as anti-aging agents based on their glycation inhibitory effect.

[0047] <High-Protein Gel Food> In one embodiment, the present invention provides a high-protein gel food in which the concentration, components, and shape of egg white (containing ovalbumin as a main component), collagen, gelatin, etc. are adjusted using CIS gel technology. Elderly people with sarcopenia or frailty need to ingest approximately 1.2 to 1.5 g / kg body weight / day or more of protein, but this is usually not easy due to the large volume. High-protein gel foods using CIS gel technology enable sufficient protein intake in a small volume.

[0048] Cultured Meat: In one embodiment, the present invention provides cultured meat obtained by culturing edible animal cells in a hydrogel (CIS gel) with a controlled concentration, composition, and shape, or a hydrate of the dried hydrogel. The colloidal solution used to prepare the CIS gel is preferably an edible one, and examples thereof include egg white (containing ovalbumin as a main component), collagen, gelatin, and agarose. Examples of animals from which edible animal cells are derived include cows, pigs, birds, horses, and fish. Examples of edible animal cells include muscle cells, adipocytes, and fibroblasts.

[0049] <<Regenerated Leather>> In one embodiment, the present invention provides a regenerated leather of a desired shape, produced by gelling a collagen solution containing crushed leather or leather produced during the leather manufacturing process using CIS gel technology, drying the gel, and then irradiating it with ultraviolet light to form crosslinks between collagen fibers derived from the leather or crushed leather produced during the leather manufacturing process and the gelled collagen fibers. This regenerated leather provides a 100% animal skin-derived regenerated leather, which is not achieved with conventional recycled leather (a sheet made by crushing leather product scraps into fibers and mixing them with latex resin, which contains approximately 40% resin and other components not derived from animal skin). Because 100% animal skin-derived regenerated leather allows for a complete recycling system that does not require combustion, it can lead to the creation of advanced decarbonization technologies for the SDGs.

[0050] The present invention will be described below with reference to examples, but the present invention is not limited to these examples. [Example 1] Approximately 20 mL of a 0.50 (w / v)% acid-soluble native collagen solution was poured into a dialysis cellulose tube (permeation molecular weight: 14,000, diameter: 16 mm) and then allowed to stand in a refrigerator. The natural concentration process of the acid-soluble native collagen solution was observed over time in the refrigerator, and the weight was measured. The results are shown in Figure 2. The weight of the acid-soluble native collagen solution immediately after pouring was 19.4 g, but after 66 hours it had decreased to 6.3 g (0.325-fold), confirming that the concentration of the acid-soluble native collagen solution was 1.54%, or approximately three times more concentrated.

[0051] [Example 2] The acid-soluble native collagen solution concentrated for 66 hours in Example 1 was immersed in a basal culture medium (DMEM) along with the dialysis tube, and the collagen sol preparation process and gelation progress were observed over time in a refrigerator. The results are shown in Figure 3. It was confirmed that uniform gel formation progressed over time, reaching a plateau after 60 minutes at the latest.

[0052] [Example 3] As shown in Figure 4, a native collagen gel prepared in a cellulose dialysis tube was immersed in purified water to remove low molecular weight components from the basal culture solution, and then the natural drying process was observed over time in a refrigerator. After confirming complete drying seven days after the start of drying, the dialysis tube was immersed in purified water to obtain a rehydrated collagen gel (collagen vitrigel-like gel). This rehydrated collagen gel could potentially be used as "artificial tendon meat."

[0053] [Example 4] As shown in Figure 5, approximately 5.0 mL of chicken egg white solution was poured into a cellulose dialysis tube (permeation molecular weight: 14,000, diameter: 6 mm). The chicken egg white solution was allowed to naturally concentrate (placed vertically) in a refrigerator for 22 hours, and then the dialysis tube was placed in a water bath at 70°C for 15 minutes. The "udon-like boiled egg white" was removed from the dialysis tube, and it was confirmed that it could be lifted with tweezers.

[0054] Comparative Example 1 As shown in Figure 6, approximately 3.0 mL of chicken egg white solution was poured into a cellulose dialysis tube (permeation molecular weight: 14,000, diameter: 6 mm), and the dialysis tube was heated in a water bath at 70°C for 20 minutes. A "soft, cylindrical boiled egg white" was removed from the dialysis tube and confirmed to be unable to be lifted with tweezers.

[0055] Example 5 As shown in Figure 7, approximately 5.0 mL of chicken egg white solution was poured into a cellulose dialysis tube (permeation molecular weight: 14,000, diameter: 6 mm). After allowing the chicken egg white solution to naturally concentrate (placed horizontally) in a refrigerator for 22 hours, the dialysis tube was placed in a 70°C water bath for 15 minutes. The dialysis tube was then naturally dried (placed horizontally) in a refrigerator. After confirming complete drying four days after the start of drying, the dialysis tube was immersed in purified water for rehydration. The "kishimen-like boiled egg white" was removed from the dialysis tube and confirmed to be able to be not only lifted but also pinched with tweezers.

[0056] Example 6 In Example 1, the liquid level of the acid-soluble native collagen solution in the dialysis cellulose tube was 106 mm immediately after injection and decreased to 38 mm after 66 hours (0.358-fold), suggesting that the change in liquid level can be used as a rough indicator of the concentration rate. Therefore, a 0.50 (w / v)% acid-soluble native collagen solution or a 1.0 (w / v)% acid-soluble atelocollagen solution was injected into a dialysis cellulose tube (molecular weight permeation: 14,000, diameter: 16 mm) and then allowed to stand in a refrigerator, and the change in liquid level was measured over time. As a result, the liquid level of approximately 20 mL of native collagen solution injected into the dialysis tube decreased from 98 mm immediately after injection to 18 mm after 5 days, 13 mm after 6 days, and 7 mm after 7 days (0.071-fold), demonstrating that the solution could be concentrated approximately 14-fold in one week. Similarly, the liquid level of approximately 100 mL of atelocollagen solution injected into a dialysis tube was 465 mm immediately after injection, 268 mm after 2 days, 129 mm after 4 days, 27 mm after 7 days, and 13 mm (0.028 times) after 9 days, demonstrating that the solution could be concentrated approximately 36 times in 9 days. Furthermore, it was confirmed that both a native collagen solution concentrated approximately 14 times (estimated concentration: approximately 7%) and an atelocollagen solution concentrated approximately 36 times (estimated concentration: approximately 36%) could form gels using the same method as in Example 2.

[0057] Example 7 Two dialysis cellulose tubes (molecular weight permeation: 14,000, diameter: 16 mm) were prepared by injecting approximately 20 mL of 0.50 (w / v)% acid-soluble native collagen solution. Both tubes were then placed in a refrigerator to concentrate the solution approximately three-fold. A plastic rod with an outer diameter of approximately 5 mm was inserted into one dialysis tube, and a rectangular mesh (MS-60 mesh, 12.5 x 77 mm) was inserted into the other dialysis tube. The dialysis tubes were then immersed in basal culture medium (DMEM) to form gels. Next, the plastic rod-containing collagen gel and mesh-containing collagen gel prepared in the dialysis tubes were immersed in purified water and then air-dried in a refrigerator. After confirming complete dryness, the dialysis tubes were immersed in purified water for rehydration, and the dialysis tubes were removed to obtain plastic rod-containing collagen vitrigel-like gels and mesh-containing collagen vitrigel-like gels. Further, the plastic rod was removed to obtain tubular collagen vitrigel-like gels (see Figure 8).

[0058] Example 8 Frozen egg white (HV) (Kewpie Egg Corporation) was thawed and then poured into three cellulose dialysis tubes (molecular weight permeation: 14,000, diameter: 6 mm) at approximately 16 mL each. All three tubes were then suspended vertically in a refrigerator and allowed to stand for 22 hours for concentration. The liquid level changed as shown in the table below.

[0059]

[0060] After 22 hours, all three tubes were transferred to Ziploc® (Daiso Industries, Large Freezer Bag) bags containing 500 mL of purified water, and the sealed Ziploc® bags were heated in an 85°C water bath for 15 minutes to denature the egg whites, producing an egg white gel. The "udon-like egg white gel" was then removed from the dialysis tube and confirmed to be able to be picked up with disposable chopsticks (see Figure 9).

[0061] Example 9 As shown in Figure 10, tannin-tanned leather was finely shredded with scissors and then sieved through a polyethylene mesh (mesh: 20, opening: 925 μm). 0.2 g of the sieved ground leather was suspended in 5 mL of 0.50 (w / v)% acid-soluble native collagen solution or 1.0 (w / v)% acid-soluble atelocollagen solution and poured into a dialysis cellulose tube (molecular weight permeation: 14,000, flat width: 25 mm). Both ends were sealed to a length of approximately 8 cm to prevent air intrusion, and the dialysis tube was immersed in basal culture medium (DMEM) to form a gel. The pulverized leather-containing collagen gel produced in the dialysis tube was then immersed in purified water to remove low-molecular-weight components from the basal culture medium, followed by air drying. After confirming complete drying, the dialysis tube was immersed in purified water to rehydrate, and the dialysis tube was then removed to obtain a pulverized leather-containing collagen vitrigel-like gel. After the crushed leather-containing collagen vitrigel-like gel was naturally dried, both the front and back surfaces of the crushed leather-containing collagen vitrigel-like gel dried body were irradiated with ultraviolet light (400 mJ / cm ２ / one side x 2 times) to obtain regenerated leather (dried native collagen vitrigel-like gel containing pulverized leather and dried atelocollagen vitrigel-like gel containing pulverized leather).

[0062] Example 10 As shown in Figure 11, 5 mL of 1.0 (w / v)% acid-soluble atelocollagen solution was poured into a regenerated cellulose dialysis tube (molecular weight permeation: 50,000, flat width: 34 mm). Both ends were sealed to a length of approximately 5 cm to prevent air intrusion, and the dialysis tube was immersed in a saline solution (110 mM NaCl, 11 mM NaHCO3) to form a gel. The plate-shaped atelocollagen gel formed in the dialysis tube was then immersed in purified water to remove low-molecular-weight components from the saline solution, followed by natural drying with the dialysis tube or natural drying with the dialysis tube removed. When the dialysis tube was naturally dried, the dialysis tube was immersed in purified water after confirming complete dryness, and then the dialysis tube was removed to obtain a membranous atelocollagen vitrigel-like gel, which was then naturally dried. The method of natural drying with the dialysis tube yielded a dried membranous atelocollagen vitrigel-like gel. The method of natural drying with the dialysis tube yielded a dried membranous atelocollagen vitrigel-like gel. The method of natural drying with the dialysis tube yielded a dried membranous atelocollagen gel. Similar results were obtained when a basal culture medium (DMEM) was used instead of the saline solution.

[0063] Example 11 As shown in Figure 12, 50 mL of basal culture medium (DMEM) was poured into a dialysis cellulose tube (molecular weight permeation: 14,000, diameter: 16 mm) with the lower end ligated, along with a weight, and the upper end was sealed. The dialysis tube was then inserted into a 50 mL graduated cylinder previously filled with 25 mL of 0.50 (w / v)% acid-soluble native collagen solution, and gelation of the collagen solution was observed over time. The collagen solution in the graduated cylinder gelled over time, reaching a plateau in macroscopic turbidity after 2 hours. After one day, the dialysis tube was removed, and it was confirmed that a cylindrical collagen gel had been formed in the graduated cylinder. This collagen gel could be easily extracted into purified water.

[0064] Example 12 As shown in Figure 13, the Thermo Scientific Slide-A-Lyzer Mini dialysis device, designed for 15 mL conical tubes, is a polypropylene cup with a regenerated cellulose membrane (molecular weight permeation: 20,000 (purple) and 3,500 (red)) attached to the bottom of the polypropylene tube. After 0.1 mL of 0.50 (w / v)% acid-soluble native collagen solution was poured into the polypropylene cup, the polypropylene cup was placed in a 35 mm diameter culture dish pre-filled with 2 mL of saline solution (110 mM NaCl, 11 mM NaHCO3) so that the regenerated cellulose membrane on the bottom of the polypropylene cup was immersed. The gelation of the collagen solution in the polypropylene cup progressed over time, and the macroscopic turbidity of the regenerated cellulose membrane reached a plateau after 2 hours for both regenerated cellulose membranes. Therefore, it was concluded that the collagen solution could be used as an insert-type 0.5% collagen gel-overlaid regenerated cellulose membrane culture carrier.

[0065] Example 13: The ad-MED Vitrigel 2 (12-well) manufactured by Kanto Chemical Co., Inc. is an insert-type culture vessel with a dried collagen vitrigel membrane attached to the bottom of a plastic tube. As shown in Figure 14, 0.1 mL of a 0.50 (w / v)% acid-soluble native collagen solution was poured into this ad-MED Vitrigel 2, and then the ad-MED Vitrigel 2 was placed in a well of a 12-well plate into which 1 mL of basal culture medium (DMEM) had been poured in advance, so that the collagen vitrigel membrane on the bottom of the ad-MED Vitrigel 2 was immersed. The gelation of the collagen solution in the ad-MED Vitrigel 2 progressed over time, and the macroscopic turbidity observation on the collagen vitrigel membrane reached a plateau after 2 hours. Therefore, it was determined that the ad-MED Vitrigel 2 could be used as an insert-type 0.5% collagen gel-overlaid collagen vitrigel membrane culture carrier.

[0066] Example 14 As shown in Figure 15, 0.8 mL of a saline solution (110 mM NaCl, 11 mM NaHCO3) was injected into ad-MED vitrigel 2 (12 wells) manufactured by Kanto Chemical Co., Inc., and then the ad-MED vitrigel 2 was placed in the wells of a 12-well plate into which 0.8 mL of a 0.50 (w / v)% acid-soluble native collagen solution had been previously injected, so that the collagen vitrigel membrane on the bottom of the ad-MED vitrigel 2 was immersed, and gelation of the collagen solution was observed over time. As a result, gelation of the collagen solution in the wells progressed over time, and the macroscopic turbidity observation reached a plateau after 2 hours directly below the collagen vitrigel membrane and after 20 hours in the surrounding area other than directly below the collagen vitrigel membrane; therefore, the ad-MED vitrigel 2 was removed from the wells. Since collagen gel formation was confirmed by visual observation in the wells from which the ad-MED vitrigel 2 had been removed, it was determined that the wells could be used as a 12-well plate-type 0.5% collagen gel culture carrier.

[0067] Example 15: The 0.5% native collagen gel in the polypropylene cup with the regenerated cellulose membrane (permeation molecular weight: 3,500) prepared in Example 12 attached thereto was placed in the well of a 12-well plate to be used as an insert-type 0.5% collagen gel-layered regenerated cellulose membrane culture carrier. 1 mL of DMEM containing 100 units / mL penicillin and 100 μg / mL streptomycin (hereinafter referred to as DMEM / PS) was poured into the well and 0.5 mL into the polypropylene cup, and the plates were allowed to stand. After approximately 1 hour, the DMEM / PS was removed. Subsequently, 1 mL of 10% FBS-containing DMEM / PS (hereinafter referred to as 10% serum-containing culture medium) was poured into the well, and C2C12 cells (mouse myoblast cell line, RIKEN Cell Bank cell number: RCB0987) were cultured in the polypropylene cup at a density of 4 × 10 ４ / cm ２ After injecting 0.5 mL of a cell suspension suspended in 10% serum-containing culture medium, the cells were incubated at 37°C in 5% CO ２Culture was initiated in an incubator. The culture medium was then replaced on days 2, 4, and 6 of culture. The culture medium replacement on days 4 and 6 was replaced with 5% FBS-containing DMEM / PS (hereinafter referred to as 5% serum-containing culture medium). As shown in Figure 16, C2C12 cells were observed over time using a phase-contrast microscope. The results showed that C2C12 cells adhered well within 3 hours of culture, and after 1 day, they showed good spreading and proliferation, reaching confluence after 7 days.

[0068] Example 16 Using a saline solution supplemented with glucose according to the preparation method of Example 13, 0.5% native collagen gels were prepared by glycation over four days at four different glucose concentrations (0, 1, 4.5, and 45 g / L) on a dried collagen vitrigel membrane in ad-MED vitrigel 2. The 0.5% collagen gel-layered collagen vitrigel membranes glycated at each glucose concentration were placed in the wells of a 12-well plate for use as insert-type culture carriers. 1 mL of DMEM / PS was poured into the well and 0.5 mL into ad-MED vitrigel 2, and the plates were allowed to stand. After approximately 1 hour, the DMEM / PS was removed. Subsequently, 1 mL of 5% serum-containing culture medium was poured into the well, and C2C12 cells (previously conditioned in 5% serum-containing culture medium) were seeded at 4 x 10 cells per well on the culture carrier in the 12-well plate. ４ / cm ２ After injecting 0.5 mL of a cell suspension suspended in 5% serum-containing culture medium, the cells were incubated at 37°C in 5% CO ２The cells were cultured in an incubator for 7 days. The culture medium was replaced on days 2, 4, and 6. The culture medium replacement on days 4 and 6 was replaced with DMEM / PS containing 1% FBS (hereinafter referred to as 1% serum-containing culture medium). As a result, as shown in Figure 17, phase-contrast microscopy revealed that some C2C12 cells cultured on collagen vitrigel membrane culture supports overlaid with 0.5% collagen gel glycated at glucose concentrations of 4.5 g / L and 45 g / L infiltrated into the collagen gel and exhibited elongated cell morphology, suggesting that the culture supports have the ability to induce C2C12 cells to infiltrate and differentiate into myocyte-like cells. In addition, a similar experiment was performed using only the collagen vitrigel membrane in ad-MED vitrigel 2 that had been glycated for 4 days at four different glucose concentrations (0, 1, 4.5, and 45 g / L). As a result, no invasion of C2C12 cells into the collagen vitrigel membrane was confirmed by observation with a phase contrast microscope of C2C12 cells cultured on the collagen vitrigel membrane that had been glycated at any of the glucose concentrations.

[0069] [Example 17] To use the 12-well plate-type 0.5% collagen gel culture carrier prepared in Example 14, 0.8 mL of DMEM / PS was poured into each well and allowed to stand. After about 1 hour, the DMEM / PS was removed. Subsequently, 1 × 10 C2C12 cells were cultured on the culture carrier. ５ / cm ２ After injecting 1 mL of a cell suspension suspended in 5% serum-containing culture medium, the cells were incubated at 37°C in 5% CO ２ Culture was initiated in an incubator. As shown in Figure 18, C2C12 cells were observed over time using a phase-contrast microscope. The results showed that C2C12 cells adhered and spread well within four hours of culture, and reached confluence after two days.

[0070] According to the present invention, it is possible to provide a basic technology for preparing a collagen gel or the like of any concentration and shape, a collagen gel or the like containing a limited molecular weight fraction component in any solution, and a basic technology for preparing said gel.

[0071] 1... cylindrical portion, 2... flange portion, 3... separation membrane, 10... culture insert type container.

Claims

1. A method for manufacturing a hydrogel or a dried hydrogel of a desired shape, comprising: Step 1 of injecting a colloidal solution into a container of a predetermined shape having a separation membrane at least partially therein; and Step 2 of placing the container in a liquid phase or a gas phase and gelling the colloidal solution in the container to obtain a hydrogel.

2. The manufacturing method according to claim 1, further comprising, between Step 1 and Step 2, Step 3 of adjusting the concentration by evaporating the moisture in the colloidal solution through the separation membrane in the container in the gas phase.

3. The manufacturing method according to claim 1, further comprising, between Step 1 and Step 2, Step 3 of adjusting the concentration by dehydrating the moisture in the colloidal solution through the separation membrane by immersing at least a part of the separation membrane of the container in a liquid phase containing a salt.

4. The manufacturing method according to claim 1, wherein the separation membrane is a dialysis membrane, an ultrafiltration membrane, a microfiltration membrane, a collagen vitrigel membrane, or a food casing.

5. The manufacturing method according to claim 1, wherein an insoluble substance of an arbitrary shape is placed in the colloidal solution and then gelled to prepare a hydrogel containing the insoluble substance.

6. The manufacturing method according to claim 5, wherein the insoluble substance is leather or crushed skin in a leather manufacturing process.

7. The manufacturing method according to claim 5, wherein the shape of the hydrogel is induced by removing the insoluble substance after gelling.

8. The manufacturing method according to claim 1, wherein gel formation is induced by diffusing, permeating, or transmitting a gelling agent in the liquid phase through the separation membrane.

9. The manufacturing method according to claim 1, wherein gel formation is induced by a temperature change in the liquid phase or the gas phase.

10. The manufacturing method according to claim 1, wherein the separation membrane is a dialysis membrane, an ultrafiltration membrane, or a microfiltration membrane, has pores for diffusing, permeating, or transmitting molecules having a molecular weight equal to or less than the allowable molecular weight, and in Step 2, a fraction component having a limited molecular weight in the liquid phase diffuses, permeates, or transmits into the colloidal solution or the hydrogel through the pores.

11. The manufacturing method according to claim 1, further comprising Step 4 of placing the container in the gas phase and evaporating the moisture of the hydrogel obtained in Step 2 through the separation membrane to concentrate the concentration of the hydrogel or obtain a dried hydrogel.

12. The manufacturing method according to any one of claims 1 to 11, wherein the container of the predetermined shape is a culture insert type container.

13. A method for producing a hydrogel or a dried hydrogel of a desired shape, comprising: Step 5 of injecting a gelling agent solution into a first container of a predetermined shape having a separation membrane at least partially therein; and after injecting a colloidal solution into a second container of a predetermined shape, bringing the gelling agent solution in the first container into contact with the colloidal solution in the second container through the separation membrane in the first container to gel the colloidal solution to obtain a hydrogel. A method for producing a hydrogel or a dried hydrogel.

14. The production method according to claim 13, further comprising Step 7 of adjusting the concentration by evaporating the moisture in the colloidal solution in the second container in the gas phase between Step 5 and Step 6.

15. A hydrogel or a dried hydrogel having uniformly colloidal particles and a limited molecular weight fraction component.

16. The hydrogel or the dried hydrogel according to claim 15, which is a collagen gel, a gelatin gel, an egg-derived gel, or an agarose gel.

17. The hydrogel or the dried hydrogel according to claim 15, wherein the limited molecular weight fraction component includes a component derived from a cell culture solution, a component derived from a conditioned culture solution, a component derived from a body fluid, a food component, or an active cosmetic ingredient.

18. A collagen disease model or a collagen aging model having uniformly collagen and advanced glycation end products.

19. Cultured meat containing a culture of a hydrate of a hydrogel or a dried hydrogel and cells of an edible part of an animal.

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